Red blood cells (RBCs) are a major diagnostic tool for important clinical data, as the metabolites and proteins within RBCs act as biomarkers to reveal numerous diseases, metabolic functions, nutritional status, and toxic metal exposure. While the proteomic study of the RBC has flourished, the metabolomic study of the RBC is still in its infancy. Among the critical metabolites required by RBC for proper function are the metals/metalloids that act as activating or structural cofactors for every class of cellular macromolecule Assessing RBC metal content is a standard clinical test, yet reference values for many of the trace metals and metalloids in the RBC are surprisingly not well known. Additionally, many metals/metalloids that are thought to be functionally relevant do not have identified cognate binding proteins, and thus their mechanisms; of action remain obscure. Therefore, establishing complete reference ranges for all metals/ metalloids (i.e. the metallome) and catalog of all metal/metalloid-binding proteins (i.e. the metalloproteome) within the RBC is essential for full clinical utility. Until now, technical obstacles prevented progress towards these goals, but we have recently developed technology that combines ultrasensitive elemental analysis with high-throughput proteomic tools, resulting in the capacity to separate and identify hundreds of proteins and to subsequently reveal bound metals/metalloids. The overall goal of this proposal is to (Aim 1) define the human RBC metallome and (Aim 2) define the human RBC metalloproteome. Mature RBCs from healthy human donors will be analyzed in whole and subcellular fractions. Proteins will be separated in liquid phase by isoelectric point and molecular weight and then identified by MALDI-TOF analysis and database matching. Elemental analysis spanning nearly the entire periodic table will be conducted to part per trillion levels in RBCs and part per million levels in separated proteins by combination of ICP and STIM/PIXE. The metalloproteome will be determined by merging proteomic and elemental maps. Results will be summarized and published in an on-line searchable database for the scientific community. This work will ultimately help integrate RBC proteomics and metabolomics for a better understanding of the role of RBCs in whole body metal homeostasis. This work will also provide the necessary foundation for larger studies to determine the effects of disease states, suboptimal nutrition, and toxic metal exposures on the RBC metallome and metalloproteome, which are likely to improve clinical diagnostics and reveal new therapeutic targets.